Actin dynamics critically regulate numerous aspects of cellular function. The proteins mediating actin turnover have been characterized in great biochemical detail. However, how these biochemical properties play out inside a live primary cell to control actin dynamics is poorly understood. For such an understanding we need global cellular concentrations of actin regulators, as protein interactions are controlled by mass action. We need local concentrations of actin regulators, as concentrations of proteins inside a live cell vary substantially in time and space and as actin dynamics are regulated locally not globally. We need diffusion constants of actin regulators, as they will influence how local concentrations change over time. These three datasets we allow the generation of predictive mathematical models that describe how actin dynamics are regulated in live cells. Given the pervasive nature of actin regulation throughout different cell types, such models will be of universal interest. Here we propose to develop the methods and acquire the data necessary for generating such a model in primary T cells. Well-defined spatiotemporal constraints, extensive data on upstream signaling, and high physiological relevance make T cells interesting as an experimental system to elucidate the regulation of actin dynamics in live primary cells. Actin turnover is driven by the nucleation of new actin filaments, their stabilization through capping, and their destruction through severing. As T cells predominantly rely on one actin nucleator, the Arp2/3 complex, we will investigate the Arp2/3 complex, its three activators WASP, WAVE2, and the hematopoietic cortactin homolog HS1, capping protein, the actin filament severing protein cofilin, and actin itself. We will build the quantitative foundation for a model of actin dynamics primary T cell activation in two aims: Aim 1: To determine global concentrations of actin regulators in primary T cell activation. We will use quantitative immunoblotting of T cell extracts to determine the global concentrations of actin regulators in primary T cells. We will also determine how such concentrations change when GFP- tagged versions of the regulators are co-expressed with the endogenous proteins. Aim 2: To determine local concentrations of actin regulators in primary T cell activation. We will use GFP-tagged versions of the actin regulators, spinning disk confocal microscopy, and quantitative three-dimensional image analysis to determine the spatiotemporal variations in the concentrations of the actin regulators. These data will be corroborated by staining of endogenous proteins. We will also use GFP-tagged versions of the actin regulators in FRAP experiments to determine the diffusion constants of the actin regulators inside live primary T cells. PUBLIC HEALTH RELEVANCE: T cells are the central regulatory cells of the immune system, the part of our body that fights infections and cancer and that is compromised in autoimmune disease. The regulation of T cell activation is thus critical. Yet it is difficult to understand, as it is a highly complex process involving dozens of different molecules whose activity varies in time and space. This complex T cell organization is driven by actin dynamics. Here we combine biochemical information and information contained in spatiotemporal patterns of actin regulators to elucidate how actin dynamics are regulated inside a live primary T cells upon activation.